Impacts of Quaternary climatic changes on the diversification of riverine cichlids in the lower Congo River.

Climatic and geomorphological changes during the Quaternary period impacted global patterns of speciation and diversification across a wide range of taxa, but few studies have examined these effects on African riverine fishes. The lower Congo River is an excellent natural laboratory for understanding complex speciation and population diversification processes as it is hydrologically extremely dynamic and recognized as a continental hotspot of diversity harboring many narrowly endemic species. A previous study using genome-wide SNP data highlighted the importance of dynamic hydrological regimes to the diversification and speciation in lower Congo River cichlids. However, historical climate and hydrological changes (e.g., reduced river discharge during extended dry periods) have likely also influenced ichthyofaunal diversification processes in this system. The lower Congo River offers a unique opportunity to study climate-driven changes in river discharge, given the massive volume of water from the entire Congo basin flowing through this short stretch of the river. Here, we, for the first time, investigate the impacts of paleoclimatic factors on ichthyofaunal diversification in this system by inferring divergence times and modeling patterns of gene flow in four endemic lamprologine cichlids, including the blind cichlid, Lamprologus lethops. Our results suggest that Quaternary climate changes associated with river discharge fluctuations may have impacted the diversification of species along the system. Our study, using reduced representation sequencing (2RADseq), indicates that the lower Congo River lamprologines emerged during the Early-Middle Pleistocene transition, characterized as one of the earth's major climatic transformation periods. Modeling results suggest that gene flow across populations and between species was not constant but occurred in temporally constrained pulses. We show that these results correlate with glacial-interglacial fluctuations. The current hyper-diverse fish assemblages of the lower Congo River riverscape likely reflect the synergistic effects of multiple drivers fueling complex evolutionary processes through time.

Habitat specialization predicts demographic response and vulnerability of floodplain birds in Amazonia.

The annual flooding cycle of Amazonian rivers sustains the largest floodplains on Earth, which harbour a unique bird community. Recent studies suggest that habitat specialization drove different patterns of population structure and gene flow in floodplain birds. However, the lack of a direct estimate of habitat affinity prevents a proper test of its effects on population histories. In this work, we used occurrence data, satellite images and genomic data (ultra-conserved elements) from 24 bird species specialized on a variety of seasonally flooded environments to classify habitat affinities and test its influence on evolutionary histories of Amazonian floodplain birds. We demonstrate that birds with higher specialization in river islands and dynamic environments have gone through more recent demographic expansion and currently have less genetic diversity than floodplain generalist birds. Our results indicate that there is an intrinsic relationship between habitat affinity and environmental dynamics, influencing patterns of population structure, demographic history and genetic diversity. Within the floodplains, historical landscape changes have had more severe impacts on island specialists, making them more vulnerable to current and future anthropogenic changes, as those imposed by hydroelectric dams in the Amazon Basin.

The United States dried seahorse trade: A comparison of traditional Chinese medicine and ecommerce-curio markets using molecular identification.

Tens of millions of dried seahorses (genus Hippocampus) are traded annually, and the pressure from this trade along with their life history traits (involved parental care and small migration distances and home ranges) has led to near global population declines. This and other forms of overexploitation have led to all seahorse species being listed in Appendix II under the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). The signatory nations of CITES recommended a 10-cm size limit of seahorses to ensure harvested individuals have reached reproductive maturity, and have thus had the chance to produce offspring, to maintain a more sustainable global seahorse fishery. We assessed adherence to CITES recommendations using DNA barcoding and size measurements to compare two prominent U.S. dried seahorse markets: (1) traditional Chinese medicine (TCM), and (2) non-medicinal ecommerce and coastal curio (ECC). We also estimated U.S. import abundance from CITES records. Of the nine species identified among all samples (n = 532), eight were found in the TCM trade (n = 168); composed mostly (75%) of the Indo-Pacific species Hippocampus trimaculatus, and Hippocampus spinosissimus, and the Latin American Hippocampus ingens. In contrast, ECC samples (n = 344) included 5 species, primarily juvenile Indo-Pacific Hippocampus kuda (51.5%) and the western Atlantic Hippocampus zosterae (40.7). The majority of TCM samples (85.7%) met the CITES size recommendation, in contrast to 4.8% of ECC samples. These results suggest non-size discriminatory bycatch is the most likely source of imported ECC specimens. In addition, CITES records indicate that approximately 602,275 dried specimens were imported into the U.S. from 2004-2020, but the exact species composition remains unknown as many U.S. imports records list one species or Hippocampus spp. from confiscated shipments due to difficulties in morphological identification and large numbers of individuals per shipment. Molecular identification was used to identify the species composition of confiscated shipment imports containing undesignated species, and similar to TCM, found H. trimaculatus and H. spinosissimus the most abundant. By combining DNA barcoding, size comparisons, and CITES database records, these results provide an important glimpse into the two primary dried U.S. seahorse end-markets, and may further inform the conservation status of several Hippocampus species.

Evidence of introgression, ecological divergence and adaptation in Asterias sea stars.

Hybrid zones are important windows into the evolutionary dynamics of populations, revealing how processes like introgression and adaptation structure population genomic variation. Importantly, they are useful for understanding speciation and how species respond to their environments. Here, we investigate two closely related sea star species, Asterias rubens and A. forbesi, distributed along rocky European and North American coastlines of the North Atlantic, and use genome-wide molecular markers to infer the distribution of genomic variation within and between species in this group. Using genomic data and environmental niche modelling, we document hybridization occurring between northern New England and the southern Canadian Maritimes. We investigate the factors that maintain this hybrid zone, as well as the environmental variables that putatively drive selection within and between species. We find that the two species differ in their environmental niche breadth; Asterias forbesi displays a relatively narrow environmental niche while conversely, A. rubens has a wider niche breadth. Species distribution models accurately predict hybrids to occur within environmental niche overlap, thereby suggesting environmental selection plays an important role in the maintenance of the hybrid zone. Our results imply that the distribution of genomic variation in North Atlantic sea stars is influenced by the environment, which will be crucial to consider as the climate changes.

Global determinants of insect mitochondrial genetic diversity.

Understanding global patterns of genetic diversity is essential for describing, monitoring, and preserving life on Earth. To date, efforts to map macrogenetic patterns have been restricted to vertebrates, which comprise only a small fraction of Earth's biodiversity. Here, we construct a global map of predicted insect mitochondrial genetic diversity from cytochrome c oxidase subunit 1 sequences, derived from open data. We calculate the mitochondrial genetic diversity mean and genetic diversity evenness of insect assemblages across the globe, identify their environmental correlates, and make predictions of mitochondrial genetic diversity levels in unsampled areas based on environmental data. Using a large single-locus genetic dataset of over 2 million globally distributed and georeferenced mtDNA sequences, we find that mitochondrial genetic diversity evenness follows a quadratic latitudinal gradient peaking in the subtropics. Both mitochondrial genetic diversity mean and evenness positively correlate with seasonally hot temperatures, as well as climate stability since the last glacial maximum. Our models explain 27.9% and 24.0% of the observed variation in mitochondrial genetic diversity mean and evenness in insects, respectively, making an important step towards understanding global biodiversity patterns in the most diverse animal taxon.

Riverscape genomics of cichlid fishes in the lower Congo: Uncovering mechanisms of diversification in an extreme hydrological regime.

Freshwater fishes are notably diverse, given that freshwater habitat represents a tiny fraction of the earth's surface, but the mechanisms generating this diversity remain poorly understood. Rivers provide excellent models to understand how freshwater diversity is generated and maintained across heterogeneous habitats. In particular, the lower Congo River (LCR) consists of a dynamic hydroscape exhibiting extraordinary aquatic biodiversity, endemicity, morphological and ecological specialization. Previous studies have suggested that the numerous high-energy rapids throughout the LCR form physical barriers to gene flow, thus facilitating diversification and speciation, generating ichthyofaunal diversity. However, this hypothesis has not been fully explored using genome-wide SNPs for fish species distributed across the LCR. Here, we examined four lamprologine cichlids endemic to the LCR that are distributed along the river without range overlap. Using genome-wide SNP data, we tested the hypotheses that high-energy rapids serve as physical barriers to gene flow that generate genetic divergence at interspecific and intraspecific levels, and that gene flow occurs primarily in a downstream direction. Our results are consistent with the prediction that powerful rapids sometimes act as a barrier to gene flow but also suggest that, at certain temporal and spatial scales, they may provide multidirectional dispersal opportunities for riverine rheophilic cichlid fishes. These results highlight the complexity of diversification processes in rivers and the importance of assessing such processes across different riverscapes.

Genomic scale data shows that Parastacus nicoleti encompasses more than one species of burrowing continental crayfishes and that lineage divergence occurred with and without gene flow.

Delimiting species is a challenge, especially in scenarios of diversification with gene flow and when species are now allopatric where reproductive isolation cannot be directly tested. Continental burrowing crayfishes of the genus Parastacus present a disjoint distribution in southern South America. One of the species is P. nicoleti, which lives in underground waters in swampy and wooded areas of southern Chile. A previous assessment based on mitochondrial DNA sequences suggest that the taxon may represent a species complex. Here, using thousands of nuclear genomic single-nucleotide polymorphisms obtained via RADSeq from 81 specimens collected at 27 localities throughout the distributional range of the species, we apply an integrative species delimitation approach to test species boundaries and to investigate some aspects of the speciation process. Our analyses corroborate previous results; a scenario that we favor suggests that the P. nicoleti encompasses seven distinct species. Additionally, demographic analyses show that the distinct species have followed distinct trajectories in size change during the last 17.5 million years and that speciation in this group occurred both in strict isolation as well as in the presence of gene flow.

A multidisciplinary framework for biodiversity prediction in the Brazilian Atlantic Forest hotspot / Integrando disciplinas para a predição da biodiversidade da Floresta Atlântica no Brasil

Abstract: We briefly describe selected results from our thematic project focused on the biodiversity of the Atlantic Forest ("AF BIOTA"), which was jointly funded by FAPESP's BIOTA Program, the U.S. National Science Foundation Dimensions of Biodiversity Program, and the National Aeronautics and Space Administration (NASA). As one of the five most important hotspots of biodiversity in the world, the Atlantic Forest (AF) holds less than 16% of its vegetation cover, yet, amongst the hotspots, it still harbors one of the highest numbers of species, including endemics. By gathering specialists across multiple disciplines (biology, geology, engineering), we aimed to understand how this megabiodiversity was built through time, informing biodiversity science and conservation. Among the results, we trained 18 Master's and 26 Ph.D. students, published more than 400 peer-reviewed papers that improved our knowledge about the forest's biologic and climatic diversity and dynamics through time, developed new analytical methods, produced outreach videos and articles, and provided data to help define biodiversity conservation policies.

Resumo: Descrevemos de forma resumida resultados selecionados do nosso projeto temático com foco na biodiversidade da Floresta Atlântica ("AF BIOTA"), que foi financiado pelo BIOTA FAPESP e pelo programa "Dimensions of Biodiversity" da "U.S. National Science Foundation" e "National Aeronautics and Space Administration" (NASA). Devido à sua megabiodiversidade (que inclui várias espécies endêmicas), e por restar menos de 16% da vegetação original, a Floresta Atlântica (FA) é uma das cinco áreas mais importantes para a biodiversidade do planeta ("biodiversity hotspot"). Reunimos especialistas de diversas disciplinas (biologia, geologia, engenharia) visando compreender como essa megabiodiversidade evoluiu ao longo do tempo e fornecer informações científicas para a sua conservação. Dentre os resultados obtidos, nós formamos 18 mestres e 26 doutores, publicamos mais de 400 artigos científicos que aumentaram o conhecimento sobre a diversidade biológica e climática da FA e sua dinâmica ao longo do tempo, desenvolvemos novos métodos analíticos, produzimos material de divulgação científica e fornecemos dados para desenvolver políticas públicas de conservação da biodiversidade.

A unified model of species abundance, genetic diversity, and functional diversity reveals the mechanisms structuring ecological communities.

Biodiversity accumulates hierarchically by means of ecological and evolutionary processes and feedbacks. Within ecological communities drift, dispersal, speciation, and selection operate simultaneously to shape patterns of biodiversity. Reconciling the relative importance of these is hindered by current models and inference methods, which tend to focus on a subset of processes and their resulting predictions. Here we introduce massive ecoevolutionary synthesis simulations (MESS), a unified mechanistic model of community assembly, rooted in classic island biogeography theory, which makes temporally explicit joint predictions across three biodiversity data axes: (i) species richness and abundances, (ii) population genetic diversities, and (iii) trait variation in a phylogenetic context. Using simulations we demonstrate that each data axis captures information at different timescales, and that integrating these axes enables discriminating among previously unidentifiable community assembly models. MESS is unique in generating predictions of community-scale genetic diversity, and in characterizing joint patterns of genetic diversity, abundance, and trait values. MESS unlocks the full potential for investigation of biodiversity processes using multidimensional community data including a genetic component, such as might be produced by contemporary eDNA or metabarcoding studies. We combine MESS with supervised machine learning to fit the parameters of the model to real data and infer processes underlying how biodiversity accumulates, using communities of tropical trees, arthropods, and gastropods as case studies that span a range of data availability scenarios, and spatial and taxonomic scales.

Rugged relief and climate promote isolation and divergence between two neotropical cold-associated birds.

The role of historical factors in establishing patterns of diversity in tropical mountains is of interest to understand the buildup of megadiverse biotas. In these regions, the historical processes of range fragmentation and contraction followed by dispersal are thought to be mediated by the interplay between rugged relief (complex topography) and climate fluctuations and likely explain most of the dynamics of diversification in plants and animals. Although empirical studies addressing the interaction between climate and topography have provided invaluable insights into population divergence and speciation patterns in tropical montane organisms, a more detailed and robust test of such processes in an explicit spatio-temporal framework is still lacking. Consequently, our ability to gain insights into historical range shifts over time and the genomic footprint left by them is limited. Here, we used niche modeling and subgenomic population-level datasets to explore the evolution of two species of warbling finches (genus Microspingus) disjunctly distributed across the Montane Atlantic Forest, a Neotropical region with complex geological and environmental histories. Population structure inferences suggest a scenario of three genetically differentiated populations, which are congruent with both geography and phenotypic variation. Demographic simulations support asynchronous isolation of these populations as recently as ∼40,000 years ago, relatively stable population sizes over recent time, and past gene flow subsequent to divergence. Throughout the last 800,000 years, niche models predicted extensive expansion into lowland areas with increasing overlap of species distributions during glacial periods, with prominent retractions and isolation into higher altitudes during interglacials, which are in line with signs of introgression of currently isolated populations. These results support a dual role of cyclical climatic changes: population divergence and persistence in mountain tops during warm periods followed by periods of expansion and admixture in lower elevations during cold periods. Our results underscore the role of the interplay between landscape and climate as an important mechanism in the evolution of the Neotropical montane biota.

Asymmetrical gene flow in five co-distributed syngnathids explained by ocean currents and rafting propensity.

Ocean circulation driving macro-algal rafting is believed to serve as an important mode of dispersal for many marine organisms, leading to predictions on population-level genetic connectivity and the directionality of effective dispersal. Here, we use genome-wide single nucleotide polymorphism data to investigate whether gene flow directionality in two seahorses (Hippocampus) and three pipefishes (Syngnathus) follows the predominant ocean circulation patterns in the Gulf of Mexico and northwestern Atlantic. In addition, we explore whether gene flow magnitudes are predicted by traits related to active dispersal ability and habitat preference. We inferred demographic histories of these co-distributed syngnathid species, and coalescent model-based estimates indicate that gene flow directionality is in agreement with ocean circulation data that predicts eastward and northward macro-algal transport. However, the magnitude to which ocean currents influence this pattern appears strongly dependent on the species-specific traits related to rafting propensity and habitat preferences. Higher levels of gene flow and stronger directionality are observed in Hippocampus erectus, Syngnathus floridae and Syngnathus louisianae, which closely associated with the pelagic macro-algae Sargassum spp., compared to Hippocampus zosterae and the Syngnathus scovelli/Syngnathus fuscus sister-species pair, which prefer near shore habitats and are weakly associated with pelagic Sargassum. This study highlights how the combination of population genomic inference together with ocean circulation data can help explain patterns of population structure and diversity in marine ecosystems.

Comparative phylogeographic inference with genome-wide data from aggregated population pairs.

Comparing divergences across multiple sister population pairs has been a focus in phylogeography since its inception. Initial approaches used organelle genetic data and involved qualitative comparisons of phylogenetic patterns to evaluate hypotheses of shared and variable evolutionary responses. This endeavor has progressed with coalescent model-based statistical techniques and advances in next-generation sequencing, yet there remains a need for methods that can exploit aggregated genomic-scale data within a unified analytical framework. To this end, we introduce the aggregate joint site frequency spectrum (ajSFS) by validating its use within a hierarchical Bayesian framework through several in silico experiments. Subsequently, we applied our method against two published restriction site-associated DNA marker datasets consisting of eight local replicates of a lamprey species pair and six co-distributed passerine taxon pairs, respectively, with the aim of inferring variability in co-divergence and co-migration histories. We found that the lamprey population pairs exhibited temporal synchrony in both co-divergence and collective secondary contact times, yet an idiosyncratic pattern in secondary migration intensities. In contrast, the bird population pairs displayed thoroughly asynchronous co-divergence histories. Our results demonstrate that the ajSFS can be exploited for complex and flexible co-demographic inference, opening up new possibilities for comparative phylogeography and population genomic studies.

Quaternary climate changes as speciation drivers in the Amazon floodplains.

The role of climate as a speciation driver in the Amazon has long been discussed. Phylogeographic studies have failed to recover synchronous demographic responses across taxa, although recent evidence supports the interaction between rivers and climate in promoting speciation. Most studies, however, are biased toward upland forest organisms, while other habitats are poorly explored and could hold valuable information about major historical processes. We conducted a comparative phylogenomic analysis of floodplain forest birds to explore the effects of historical environmental changes and current connectivity on population differentiation. Our findings support a similar demographic history among species complexes, indicating that the central portion of the Amazon River basin is a suture zone for taxa isolated across the main Amazonian sub-basins. Our results also suggest that changes in the fluvial landscape induced by climate variation during the Mid- and Late Pleistocene drove population isolation, leading to diversification with subsequent secondary contact.

ABLE: blockwise site frequency spectra for inferring complex population histories and recombination.

We introduce ABLE (Approximate Blockwise Likelihood Estimation), a novel simulation-based composite likelihood method that uses the blockwise site frequency spectrum to jointly infer past demography and recombination. ABLE is explicitly designed for a wide variety of data from unphased diploid genomes to genome-wide multi-locus data (for example, RADSeq) and can also accommodate arbitrarily large samples. We use simulations to demonstrate the accuracy of this method to infer complex histories of divergence and gene flow and reanalyze whole genome data from two species of orangutan. ABLE is available for download at https://github.com/champost/ABLE .

Differential Effect of Selection against LINE Retrotransposons among Vertebrates Inferred from Whole-Genome Data and Demographic Modeling.

Variation in LINE composition is one of the major determinants for the substantial size and structural differences among vertebrate genomes. In particular, the larger genomes of mammals are characterized by hundreds of thousands of copies from a single LINE clade, L1, whereas nonmammalian vertebrates possess a much greater diversity of LINEs, yet with orders of magnitude less in copy number. It has been proposed that such variation in copy number among vertebrates is due to differential effect of LINE insertions on host fitness. To investigate LINE selection, we deployed a framework of demographic modeling, coalescent simulations, and probabilistic inference against population-level whole-genome data sets for four model species: one population each of threespine stickleback, green anole, and house mouse, as well as three human populations. Specifically, we inferred a null demographic background utilizing SNP data, which was then exploited to simulate a putative null distribution of summary statistics that was compared with LINE data. Subsequently, we applied the inferred null demographic model with an additional exponential size change parameter, coupled with model selection, to test for neutrality as well as estimate the strength of either negative or positive selection. We found a robust signal for purifying selection in anole and mouse, but a lack of clear evidence for selection in stickleback and human. Overall, we demonstrated LINE insertion dynamics that are not in accordance to a mammalian versus nonmammalian dichotomy, and instead the degree of existing LINE activity together with host-specific demographic history may be the main determinants of LINE abundance.

Phenotypic and Genetic Structure Support Gene Flow Generating Gene Tree Discordances in an Amazonian Floodplain Endemic Species.

Before populations become independent evolutionary lineages, the effects of micro evolutionary processes tend to generate complex scenarios of diversification that may affect phylogenetic reconstruction. Not accounting for gene flow in species tree estimates can directly impact topology, effective population sizes and branch lengths, and the resulting estimation errors are still poorly understood in wild populations. In this study, we used an integrative approach, including sequence capture of ultra-conserved elements (UCEs), mtDNA Sanger sequencing and morphological data to investigate species limits and phylogenetic relationships in face of gene flow in an Amazonian endemic species (Myrmoborus lugubris: Aves). We used commonly implemented species tree and model-based approaches to understand the potential effects of gene flow in phylogenetic reconstructions. The genetic structure observed was congruent with the four recognized subspecies of M. lugubris. Morphological and UCEs data supported the presence of a wide hybrid zone between M. l. femininus from the Madeira river and M. l. lugubris from the Middle and lower Amazon river, which were recovered as sister taxa by species tree methods. When fitting gene flow into simulated demographic models with different topologies, the best-fit model indicated these two taxa as non-sister lineages, a finding that is in agreement with the results of mitochondrial and morphological analyses. Our results demonstrated that failing to account for gene flow when estimating phylogenies at shallow divergence levels can generate topological uncertainty, which can nevertheless be statistically well supported, and that model testing approaches using simulated data can be useful tools to test alternative phylogenetic hypotheses.

Strategies for improving approximate Bayesian computation tests for synchronous diversification.

BACKGROUND: Estimating the variability in isolation times across co-distributed taxon pairs that may have experienced the same allopatric isolating mechanism is a core goal of comparative phylogeography. The use of hierarchical Approximate Bayesian Computation (ABC) and coalescent models to infer temporal dynamics of lineage co-diversification has been a contentious topic in recent years. Key issues that remain unresolved include the choice of an appropriate prior on the number of co-divergence events (Ψ), as well as the optimal strategies for data summarization. METHODS: Through simulation-based cross validation we explore the impact of the strategy for sorting summary statistics and the choice of prior on Ψ on the estimation of co-divergence variability. We also introduce a new setting (ß) that can potentially improve estimation of Ψ by enforcing a minimal temporal difference between pulses of co-divergence. We apply this new method to three empirical datasets: one dataset each of co-distributed taxon pairs of Panamanian frogs and freshwater fishes, and a large set of Neotropical butterfly sister-taxon pairs. RESULTS: We demonstrate that the choice of prior on Ψ has little impact on inference, but that sorting summary statistics yields substantially more reliable estimates of co-divergence variability despite violations of assumptions about exchangeability. We find the implementation of ß improves estimation of Ψ, with improvement being most dramatic given larger numbers of taxon pairs. We find equivocal support for synchronous co-divergence for both of the Panamanian groups, but we find considerable support for asynchronous divergence among the Neotropical butterflies. CONCLUSIONS: Our simulation experiments demonstrate that using sorted summary statistics results in improved estimates of the variability in divergence times, whereas the choice of hyperprior on Ψ has negligible effect. Additionally, we demonstrate that estimating the number of pulses of co-divergence across co-distributed taxon-pairs is improved by applying a flexible buffering regime over divergence times. This improves the correlation between Ψ and the true variability in isolation times and allows for more meaningful interpretation of this hyperparameter. This will allow for more accurate identification of the number of temporally distinct pulses of co-divergence that generated the diversification pattern of a given regional assemblage of sister-taxon-pairs.

multi-dice: r package for comparative population genomic inference under hierarchical co-demographic models of independent single-population size changes.

Population genetic data from multiple taxa can address comparative phylogeographic questions about community-scale response to environmental shifts, and a useful strategy to this end is to employ hierarchical co-demographic models that directly test multi-taxa hypotheses within a single, unified analysis. This approach has been applied to classical phylogeographic data sets such as mitochondrial barcodes as well as reduced-genome polymorphism data sets that can yield 10,000s of SNPs, produced by emergent technologies such as RAD-seq and GBS. A strategy for the latter had been accomplished by adapting the site frequency spectrum to a novel summarization of population genomic data across multiple taxa called the aggregate site frequency spectrum (aSFS), which potentially can be deployed under various inferential frameworks including approximate Bayesian computation, random forest and composite likelihood optimization. Here, we introduce the r package multi-dice, a wrapper program that exploits existing simulation software for flexible execution of hierarchical model-based inference using the aSFS, which is derived from reduced genome data, as well as mitochondrial data. We validate several novel software features such as applying alternative inferential frameworks, enforcing a minimal threshold of time surrounding co-demographic pulses and specifying flexible hyperprior distributions. In sum, multi-dice provides comparative analysis within the familiar R environment while allowing a high degree of user customization, and will thus serve as a tool for comparative phylogeography and population genomics.

Comment on Rieux and Balloux: calibration from tip-dating can compromise topological accuracy and evolutionary inference.

We contribute to the recent review of Rieux & Balloux, 2016, Mol. Ecol., 25, 1911 on inferences from tip-dated phylogenies by developing their discussion on the influence of population size (Ne ) under panmixia for the estimation of substitution rate (µ). We highlight how phylogenetic trees inferred with tip-dated sequences under large panmictic Ne tend to erroneously enforce an age-based coalescent pattern on the posterior distribution of trees, which in turn results in systematically inflated estimates of µ. We discuss the consequences of this and suggest how to accommodate the issue in the short term and long term.